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The Journal of Neuroscience, March 25, 2015 • 35(12):5067–5086 • 5067 Cellular/Molecular Characterizing KIF16B in Neurons Reveals a Novel Intramolecular “Stalk Inhibition” Mechanism That Regulates Its Capacity to Potentiate the Selective Somatodendritic Localization of Early Endosomes X Atena Farkhondeh,1 Shinsuke Niwa,1 Yosuke Takei,1 and Nobutaka Hirokawa1,2 1Department of Molecular and Cell Biology and Department of Molecular Structure and Dynamics, Graduate School of Medicine, University of Tokyo, Tokyo 113-0033, Japan, and 2Center of Excellence in Genome Medicine Research, King Abdulaziz University, Jeddah 21589, Saudi Arabia An organelle’s subcellular localization is closely related to its function. Early endosomes require localization to somatodendritic regions in neurons to enable neuronal morphogenesis, polarized sorting, and signal transduction. However, it is not known how the somatoden- dritic localization of early endosomes is achieved. Here, we show that the kinesin superfamily protein 16B (KIF16B) is essential for the correct localization of early endosomes in mouse hippocampal neurons. Loss of KIF16B induced the aggregation of early endosomes and perturbed the trafficking and functioning of receptors, including the AMPA and NGF receptors. This defect was rescued by KIF16B, emphasizing the critical functional role of the protein in early endosome and receptor transport. Interestingly, in neurons expressing a KIF16B deletion mutant lacking the second and third coiled-coils of the stalk domain, the early endosomes were mistransported to the axons. Additionally, the binding of the motor domain of KIF16B to microtubules was inhibited by the second and third coiled-coils (inhibitory domain) in an ATP-dependent manner. This suggests that the intramolecular binding we find between the inhibitory domain and motor domain of KIF16B may serve as a switch to control the binding of the motor to microtubules, thereby regulating KIF16B activity. We propose that this novel autoregulatory “stalk inhibition” mechanism underlies the ability of KIF16B to potentiate the selective somatodendritic localization of early endosomes. Key words: early endosome; KIF16B; neurons; receptor trafficking; stalk inhibition Introduction diseases, including neurodegeneration, cancer, and developmen- Neurons are highly polarized cells with a long axon and short tal defects (Salinas et al., 2008; Sarli and Giannis, 2008; Wood et dendrites (Nicholls, 2001). Commensurate with specific roles of al., 2008; Gerdes et al., 2009). The intraneuronal localization of axons and dendrites, the localization of organelles and proteins some organelles, such as mitochondria, synaptic vesicle precur- within these structures is differentially regulated. Motor proteins sors, and RNA-protein complexes, are regulated by KIFs (Nan- determine the subcellular localization of many organelles in neu- gaku et al., 1994; Okada et al., 1995; Zhao et al., 2001; Kanai et al., rons (Hirokawa et al., 2010). The kinesin superfamily proteins 2004; Cai et al., 2005; Glater et al., 2006; Niwa et al., 2008). The (KIFs) are microtubule and ATP-dependent motor proteins, appropriate localization of kinesin motors and their cargoes is most members of which transport cargoes to the microtubules critical for cell function. plus-end (Vale, 2003; Verhey and Hammond, 2009; Hirokawa et The early endosome (EE) is a membrane-bound organelle al., 2010). Defects in motor transport are associated with many that serves as a hub for receptor sorting, trafficking, signal trans- duction, and protein degradation (Hoogenraad et al., 2010; Lasiecka et al., 2010). EEA1-positive EEs are mainly localized in Received Oct. 13, 2014; revised Jan. 20, 2015; accepted Feb. 19, 2015. Author contributions: A.F., S.N., Y.T., and N.H. designed research; A.F., S.N., and Y.T. performed research; A.F. the somatodendritic region of neurons (Wilson et al., 2000). contributed unpublished reagents/analytic tools; A.F., S.N., Y.T., and N.H. analyzed data; A.F., S.N., Y.T., and N.H. When axonal proteins are missorted to dendrites, they are re- wrote the paper. moved from the dendritic plasma membrane by endocytosis, This work was supported by the Ministry of Education, Culture, Sports, Science and Technology of Japan Grant- transported to somatodendritic EEs, and re-sorted to axons in-Aid for Specially Promoted Research to N.H. We thank the members of the N.H. laboratory for assistance and valuable discussions; Y. Noda, T. Ogawa, Y. Kanai, Y. Tanaka, H. Miki, R. Nitta, H. Yajima, K. Nakajima, K. Mitsumori, (Sampo et al., 2003). However, the molecular mechanism deter- and X. Fang for technical assistance; and H. Sato, H. Fukuda, and T. Akamatsu for help. mining the specific somatodendritic localization of EEs remains The authors declare no competing financial interests. unknown. Correspondence should be addressed to Dr. Nobutaka Hirokawa, Department of Molecular and Cell Biology, KIF16B, a member of the kinesin-3 family, was identified as Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. E-mail: the plus-end motor protein that transports Rab5-positive EEs [email protected]. DOI:10.1523/JNEUROSCI.4240-14.2015 and Rab14-positive vesicles in non-neuronal cells (Hoepfner et Copyright © 2015 the authors 0270-6474/15/355067-20$15.00/0 al., 2005; Ueno et al., 2011). KIF16B has a conserved motor do- 5068 • J. Neurosci., March 25, 2015 • 35(12):5067–5086 Farkhondeh et al. • KIF16B in Neurons and “Stalk Inhibition” Mechanism Farkhondeh et al. • KIF16B in Neurons and “Stalk Inhibition” Mechanism J. Neurosci., March 25, 2015 • 35(12):5067–5086 • 5069 Here, using a variety of molecular and cell biology methods, we investigated the function of KIF16B in neurons. We show that the stalk domain of KIF16B brings a potential to determine the specific somatodendritic localization of EEs mediated by a novel intramolecular inhibitory mechanism. Materials and Methods Cloning KIF16B and construct engineering. Human KIF16B (NM_ 001199865.1) was purchased from Thermo Scientific cDNA library, and PCRed using KOD-plus DNA polymerase (Toyobo). All PCR products were cloned into pEGFP-N1, pEGFP-C1, pIRES, or pPAmCherry1-N1 (Clontech). The 3xFLAG-N1 was derived from pEGFP-N1 by replacing the GFP with a 3xFLAG tag. The 2xFYVE was generated by PCR from an HGF-regulated tyrosine kinase substrate (Riken Fantom clone Movie 1. Dynamics of colocalized KIF16B and early endosomes in neurons. KIF16B-mCherry B230365P04) to serve as an endosomal marker and was cloned into (red) and GFP-2xFYVE (green) were cotransfected into hippocampal neurons and observed miRNA, pIRES, pEGFP-C1, and the pGEX-4T1 vectors. Deletion mu- using an LSM710 Duo confocal laser-scanning microscope (Carl Zeiss). The movie shows colo- tants were made by amplifying KIF16B nucleotides corresponding to calized KIF16B and EE bidirectional movements in the somatodendritic region. The cell body of residues 1–560, 1–643, 1–810, 1–936, 1–1085, 1–1096, 1–1116, 1117– the neuron is on the right, and the dendrites are on the top left. The time-lapse covers 5 min, 1266, 548–1266, 1–810, and 1117–1266 (⌬second and third coiled- ␮ with images acquired every 2 s. Scale bars, 10 m. coils), 809–1096, and a chimera was generated using the motor domain of KIF5C, 1–560, and the PX domain, 1117–1266, of KIF16B. All con- main, three coiled-coils in the stalk domain, and a PX domain. In structs were engineered by PCR and cloned into pEGFP-N1 and HeLa cells, KIF16B regulates the recycling and degradation of pEGFP-C1 vectors. L1197A/F1198A point mutations in the PX domain EGF-receptors by controlling the localization and function of EEs of full-length K1F16B (KIF16BFull) were generated using the QuikChange II Site-Directed Mutagenesis Kit (Stratagene) and cloned in (Hoepfner et al., 2005). A structural study has shown that the PX pEGFP-N1. domain binds directly to phosphatidylinositol-3-phosphate Antibodies. Anti-KIF16B antibody was generated by immunizing (PI3P) at the endosomal membrane (Blatner et al., 2007). Intro- rabbits with the synthetic peptide (CEFSPFFKKGVFDYSSHGTG) con- ducing a point mutation that abolishes the binding to PI3P jugated with keyhole limpet hemocyanin, using an Imject maleimide- changes the localization of KIF16B. In mouse embryonic fibro- activated immunogen conjugation kit (Pierce). After four injections, blasts, KIF16B transports FGF-receptor-carrying vesicles from serum was collected and affinity-purified with antigen peptides using the Golgi to the plasma membrane via EEs (Ueno et al., 2011). SulfoLink columns (Pierce). The efficiency of the generated anti-KIF16B KIF16B is also reportedly involved in tubule formation and fis- antibody was determined using Western blotting and immunostaining. sion by inducing early endosomal fusion (Skjeldal et al., 2012). The antibodies used for immunofluorescence, Western blotting, coim- Another study showed that KIF16B mediates the transcytosis of munoprecipitation, and microtubule binding assays were as follows: anti-␤3-tubulin (Sigma, 1:1000), anti-MAP2 (Abcam, 1:100), anti- transferrin receptors to apical recycling endosomes in epithelial GM130 (BD Biosciences, 1:1000), anti-synaptotagmin (Millipore Biosci- cells (Perez Bay et al., 2013). A recent paper suggested that ence Research Reagents, 1:500), anti-EEAI (sc-6415, Santa Cruz kinesin-3 motors undergo cargo-mediated dimerization around Biotechnology, 1:100), anti-FLAG M2 (Sigma, 1:1000), anti-␣-tubulin the neck coil segment, which results in processive
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  • Normalized Expression Values *

    Normalized Expression Values *

    Normalized expression values 0.0 0.2 0.4 0.6 0.8 1.0 1.2 PLCG1 * T CD5 MP T B TCF7 cDC UBASH3A BCL11B C5AR1 * MP TLR4 CXCR5 B CD79B VPREB3 XCR1 CADM1 BEND5 * ARGHAP22 * cDC CIITA ZBTB46 FLT3 PLEKHA5 Figure S1. qPCR analysis of the core gene expression signature of cDC, MP, B and T cells in the chicken cell suBsets. RNA from cDC, MP, T and B cells of 2 disUnct pools of 4 chicken spleen was subjected to qPCR detecUon of the core gene expression signatures of immune cell subsets established in Fig. 3 and of transcripts from the mouse and human gene subset selected compendia that could not be detected on the array due to defecUve probes, i.e. ARGHAP22, BEND5, C5AR1, and PLCG1 (labeled by a star on the figure). Data are represented as the mean and SD of relave gene expression levels normalized to GAPDH expression and the maximal expression across the cell types was set to 1 (independent experimental duplicates). B cell T cell cDC Monocyte/MP Chicken Human Mouse Figure S2. Unsupervised hierarchical clustering of orthologous immune response genes across chicken, human and mouse reveals globally conserved clusters of lymphoïd- specific and myeloïd- specific genes. Heatmap of cross-normalized expression profiles for immune response genes present on all three species arrays and regulated at least 2 folds across all cell suBsets, including chicken B cells (c_B), T cells (c_CD3), MP (c_MP) and cDC (c_cDC), human B cells (h_B), T cells (h_CD4_T and h_CD8_T), monocyte-derived MP (h_MoMP), peripheral blood mononucleated cell-derived MP (h_PBMC_MP), non-classical monocytes (h_non- classical_MO), classical monocytes (h_classical_MO), BDCA3+ cDC (h_BDCA3), BDCA1+ cDC (h_BDCA1), murine B cells (m_B), T cells (m_CD4_T and m_CD8_T), peritoneal cavity MP (m_PC_MPII-480HI), lung MP (m_LU_MP), non-classical monocytes (m_non-classical_MO), classical monocytes (m_classical_MO), splenic CD8α+ cDC (m_SP_DC1), suBcutaneous lymph node CD8α+ cDC (m_LN_DC1), splenic CD11B+ cDC (m_SP_DC2), suBcutaneous lymph node CD11B+ cDC (m_LN_DC2).